Members of my lab have been studying the combined effect of activated ethanol-inducible cytochrome P450-2E1 (CYP2E1), a pro-oxidant enzyme, and suppressed mitochondrial aldehyde dehydrogenase (ALDH2), an antioxidant enzyme for the removal of toxic acetaldehyde and lipid peroxides, on causing tissue injury by alcohol and other potentially toxic substances. Alcohol-induced nitroxidative stress not only elevates the amounts of acetaldehyde and lipid peroxides but also promotes many different post-translational modifications (PTMs) of cellular proteins, contributing to mitochondrial dysfunction, ER stress and organ damage. In the past, we demonstrated the role of CYP2E1 in stimulating protein oxidation and nitration in rodent tissues. We also developed sensitive methods to specifically identify oxidatively-modified and nitrated proteins to establish their functional roles in causing tissue injury. In continuation, we have studied the role of stress-activated JNK in promoting mitochondrial dysfunction and acute liver injury by carbon tetrachloride (CCl4). To achieve this goal, we developed a sensitive method to identify its phosphorylated target proteins. Liver histology, blood alanine aminotransferase, and other enzyme activities were measured in mice exposed to a single dose of 50 mg CCl4/kg ip) without or with a highly-specific JNK inhibitor SU3327. JNK was activated within 1 h while markedly increased phosphorylation of many mitochondrial proteins observed between 1 and 8 h following CCl4 exposure. Pre-treatments with SU3327 significantly reduced phosphorylation of mitochondrial proteins at 2 h and liver damage usually observed at 24 h post-CCl4 injection. Phosphoproteins were purified from control or CCl4-exposed mice and analyzed by mass-spectrometry. Differential proteomic analysis identified many phosphorylated mitochondrial proteins involved in antioxidant defense, electron transfer, energy supply, fatty acid oxidation, etc. ALDH2, NADH-ubiquinone oxidoreductase, and alpha-ketoglutarate dehydrogenase were phosphorylated in CCl4-exposed mice but not phosphorylated in SU3327-pretreated tissues. Consistently, the suppressed activities of these enzymes in CCl4-exposed mice were restored by SU3327 pretreatment. These data provide a novel mechanism by which JNK, rapidly activated by CCl4, promotes mitochondrial dysfunction and acute hepatotoxicity through phosphorylation of numerous mitochondrial proteins. We believe that our method can be used to characterize JNK-target proteins in other tissues where JNK is activated under stressful conditions. In collaboration with Dr. Bonghee Lee at Gachon University, Korea, we also studied the role of another PTM i.e., advanced glycation end adduct with albumin (AGE-albumin) in alcohol-induced brain damage in rats. Daily oral administrations of alcohol (5 g/kg/day for 10 consecutive days) activated microglial cells to produce large amounts of AGE-albumin, which eventually caused neuronal cell death in rat hippocampus. The neuronal death by AGE-albumin was mediated through activation of JNK and p38-kinase. Increased AGE-albumin synthesis and secretion were also observed in cultured microglial cells in an ethanol concentration-dependent manner. Treatment with a soluble receptor for AGE (RAGE) or AGE inhibitors significantly reduced neuronal damage in the alcohol-exposed rats. Consistently, the elevated levels of activated microglial cells, AGE-albumin and neuronal loss were also observed in autopsied human brain specimens from alcoholic individuals compared to normal controls. These results indicated that increased AGE-albumin produced from activated microglial cells induces neuronal death, and that efficient regulation of its synthesis and secretion can be a therapeutic target for preventing alcohol-induced neurodegeneration. We recently reported the critical role of CYP2E1 in binge alcohol-mediated gut leakiness, endotoxemia, and advanced inflammatory liver disease. Binge alcohol administration (6 g/kg oral gavage for 3 times at 12-h intervals) increased the levels of serum endotoxin, hepatic enterobacterial contents, and hepatic fat accumulation with inflammatory foci in WT. The increased serum endotoxin, liver inflammation and hepatic contents of enterobacteria were significantly reduced when ethanol-exposed WT mice were co-treated with a specific CYP2E1 inhibitor chlormethiazole (CMZ) or an antioxidant N-acetylcysteine (NAC). Binge alcohol markedly elevated the levels of nitroxidative marker proteins CYP2E1 and iNOS in intestinal epithelial cells of WT mice. In contrast, these changes were not observed in the ethanol-exposed Cyp2e1-null mice or ethanol-exposed WT mice pretreated with CMZ and NAC, suggesting a critical role of CYP2E1 in gut leakiness and endotoxemia following binge alcohol exposure. In addition, similar results were observed in HIV-transgenic rats where binge alcohol (>3.5 g/kg oral gavage for 3 times at 12-h intervals) caused gut leakiness with elevated levels of serum endotoxin and pro-inflammatory cytokines and chemokines, contributing to inflammatory liver disease. The ethanol-exposed HIV-transgenic rats can be used as a surrogate model to study the mechanisms of many disease states caused by heavy alcohol intake in HIV-infected people with or without highly active anti-retroviral therapy. Based on these results, we are studying the role of different PTMs of tight junction proteins in binge alcohol-mediated gut leakiness and endotoxemia. By analyzing the tissues in different ages, we also observed that CYP2E1 is involved in aging-related liver inflammation, apoptosis and fibrosis. Elevated hepatosteatosis, ballooning degeneration, and inflammatory cell infiltration were observed in aged WT mice and these changes were accompanied with significantly higher hepatic triglyceride and serum cholesterol in aged WT compared to young mice. Aged WT also showed the highest levels of hepatocyte apoptosis and hepatic fibrosis. Further, the highest levels of hepatic hydrogen peroxide, lipid peroxidation, protein carbonylation, nitration, and oxidative DNA damage, as evidenced by the levels of 8-oxo-2'-deoxyguanosine, were observed in aged WT. However, these aging-related oxidative changes were absent or very low in aged Cyp2e1-null mice. These data suggest that CYP2E1 is important in causing aging-related hepatic steatosis, apoptosis and fibrosis possibly through increasing nitroxidative stress and that CYP2E1 can be a potential target for translational research in preventing aging-related liver disease. Based on our mechanistic studies on alcoholic fatty liver disease (AFLD) and NAFLD/NASH in our mouse strains, we performed translational research by evaluating the beneficial effects of dietary supplements including walnuts and pomegranates against AFLD and NAFLD/NASH in animal models. Our results showed that supplementation with walnuts at physiologically relevant levels (20% energy-derived) for 20 weeks did not alter body weight or visceral fat mass but significantly prevented HFD (45% energy-derived)-induced fatty liver possibly by activating AMP-kinase, a key metabolic regulator. These hepatic improvements by walnuts also coincided with reduction of HFD-induced inflammation of adipose tissues and macrophage infiltration. However, the dietary walnuts did not significantly alter HFD-induced peripheral glucose intolerance or insulin resistance despite a trend of improvement. Since 45%-energy derived HFD could be too strong to be reversed, we are considering another substance such as fructose for testing the beneficial effects of dietary supplements against insulin resistance and inflammatory tissue injury. In addition, we plan to collaborate with members of Dr. Joe Hibbelns Section at LMBB to study the beneficial effects of n-3 fatty acids on liver steatosis and obesity in rodent models.